HIV-infected individuals accumulate a reservoir of treatment-resistant, latently infected resting CD4+ T cells, even when therapy is started shortly after exposure. It remains unclear how treatment resistant HIV viral reservoirs are maintained. One theory is that latently infected cells are established early in infection and that these cells persist in the presence of HAART. A second theory is that a very low level of HIV replication is required for viral persistence. The goal of this application is to determine if ongoing replication can be demonstrated to occur in the presence of HAART. If this can be demonstrated to occur then it should be considered as a potential mechanism for reservoir maintenance. Distinguishing between these theories is important because it will drive what approaches we should take if HIV infection is to be eradicated from infected patients. However, regardless of the mechanism(s) of reservoir maintenance or the approaches to eradication of HIV, assays that detect ongoing HIV replication would be useful for monitoring therapy. In an effort to assess if ongoing replication occurs in patients on HAART, we propose to monitor total and integrated HIV DNA over time after initiating HAART. The idea behind the proposal is that in the absence of ongoing replication total DNA should eventually equal integrated HIV DNA. We will measure total and integrated HIV DNA in mononuclear cells in both blood and the GI tract and test if an excess correlates with independent measures of ongoing replication (Aim1A). In addition, we will measure these intermediates within subsets of these cells (Aim1B). Finally, we will take a complementary approach to determine if ongoing replication occurs by testing for viral spread to short lived cells (Aim1C). We expect our experiments will provide information on whether ongoing replication occurs in HIV infected individuals on HAART and potentially new information on HIV pathogenesis. PUBLIC HEALTH RELEVANCE: It is unclear why HIV is treatable, but not curable. It appears that there is a treatment resistant reservoir, but how this reservoir is maintained in the presence of HAART is unclear. In this grant proposal, we try to address this question by determining if HIV replication is completely stopped with antiviral therapy. We propose experiments that attempt to answer this question by measuring viral DNA intermediates over time on antiviral therapy.